We applied three types of computer tests to measure the dominant and non-dominant hands: the simple motor reaction time through the Human Benchmark test, the recognition time by the Hit-the-dots and the cognitive reaction time by the Trail making test part B. (3) Results: For dominant and non-dominant hands, better results of individual sports were for: simply reaction time—boxing; recognition reaction time—taekwondo; cognitive reaction—judo. (4) Conclusions: Athletes had better simple reaction with the left hand than with the right hand. Athletes had better recognition and cognitive reaction time with the right hand than with the left hand regardless of the dominant hand. The outcomes of our study indicate that the reaction times of left and right hands were influenced by the hand laterality, the type of applied stimulus, the stress complexity of tests and the type of practiced sport. Simple reaction time (SRT) is a simple reaction to a single stimulus. Recognition reaction time (RRT) involves a cognitive process of selecting the optimal response to multiple stimuli, and the response is dependent on the type and form of the stimuli. Cognitive reaction time (CRT) consists of identifying the significance of the stimuli, the association and application of knowledge in order to develop an optimal cognitive response in agreement with the stimulus complexity. Research on the hand laterality facilitated the understanding of the way of training and control: characteristics and reaction time of motor skills, responsiveness and motor coordination, thus contributing to optimizing human motor capacity. Factors that can affect the average human reaction time (RT) include: age, sex, left- or right-hand dominance, central and peripheral vision, practice, fatigue, fasting, breathing cycle, types of personality, exercise and intelligence of the subject . Reaction time components are: mental processing time, afferent nerve conduction time, movement analysis time and device response time ,3, and tComputer-based assessment of reaction time offers benefits over paper-and-pencil measures in the form of millisecond timing accuracy, reliable and randomized presentation of stimuli over multiple trials and repeated administrations and unobtrusive measurement of cognitive skills and reaction times during all aspects of the assessment process . ComputeAn innovative aspect of this study consists highlighting the interrelationships between the dominant and non-dominant hand (hand-D and hand-nonD), as well as the specific executions with the right and the left hand of the athletes in six studied sports in terms of SRT, RRT, CRT. The aim of the study was to explore the differences between simple reaction time, recognition reaction time and cognitive reaction time, between the right hand and the left hand according to the types of sport using computer games as measurement tools. The results of the study should enhance our understanding of the interrelationship between the dominant and non-dominant hand actions in optimizing sports performances.The research was conducted between March 2016 and June 2017. The dominant hand was established by asking the subjects which hand they used while performing various motor actions. The students were asked about the hand used on the computer mouse. Within the study, we applied three types of measurement tests: the simple motor reaction time (SRT) through the Human Benchmark , the recThe study aimed at assessing SRT, RRT and CRT. This cross-sectional study included 332 students specialized in physical education and sports: 187 (56.32%) from the University of Medicine and Pharmacy Targu Mures, Romania and 145 (43.68%) from Kostamonu University, Turkey. The inclusion criteria were: active athletes, participants in national and international competitions in 2016 and 2017. The subjects were randomly selected for the study. The exclusion criteria: practicing other types of individual sports than those under study; upper body injuries requiring more than one month of rest. The subjects of the study were active athletes practicing various sports at a competitive youth and senior levels individual sports practitioners (ISG). The distribution of athletes according to the practiced sport by ISG included: boxing 46 (13.9%), gymnastics 58 (17.5%), judo 64 (19.3%), karate 62 (18.7%), wrestling 48 (14.4%), taekwondo 54 (16.2%). The ISG included 278 right-handed laterality athletes (83.7%) and 54 left-handed laterality athletes (16.3%). Age characteristics of the mean age sample ± SD 20.38 ± 1.20; right handed group 20.21 ± 1.20; left handed group 20.11 ± 1.07; male group 20.42 ± 1.173; female group 19.83 ± 1.136. SRT, with the computer-gaming reaction test of Human Benchmark, was designed to evaluate the reaction time to frontal visual stimuli. The testing was carried out with dominant and non-dominant hands, five executions for each hand/2 trials and we took into consideration the average of reaction time for the best trial result in miliseconds (ms). Instructions: when the red box turns green, click as quickly as you can; five trials for each hand, counting the best average in seconds from two sessions/hand-D and hand-nonD. RRT with Hit-the-dots Reaction Test designed by University of Washington . The goaThe participants of the study were asked to perform as quickly as possible. Data from the best of two trials for both hands were used for analysis. Subjects were allowed a trial test before starting each test session. The tests were applied in different evaluation sessions with a 30 min break. The order of the tests was: SRT, RRT, CRT. The subjects were initially tested with the hand-D and then with the hand non-D. For the Benchmark test and the Hit-the-dot test we used Dell computers with CORE I7 processor of the same generation, the Hewlett-Packard mice were identical, and for the TMT test, we downloaded the application to Iphone 6s phones.p < 0.05. For parametric comparison of two groups we used t-test. For repeated measures comparisons we have used General Linear Model with the dominant arm variable as between the subjects factor. The data were processed using IBM-SPSS 20. The main statistical indicators were: mean, standard deviation (SD), mean difference (MD), partial effect size (ηp2). Descriptive statistics (mean ± SD) were calculated for all variables. The value of statistical significance was set at Our study revealed that all subjects normally use the right hands. However, some of them stated that the left hands were dominant.pvalue < 0.045, so the athletes with the right hand-D, at Benchmark test, hand-D had lower SRT performance than hand-nonD being slower by 4.75 ms. SRT to visual stimuli the differences being insignificant as follows: between right hand-D compart with left hand-nonD 8.37 ms; between left hand-D and right hand-nonD −13.91 ms; between right-left hands-D 9.03 ms; between right-left hands-nonD −13.25 ms. For ISG hand-D they perform faster than hand-nonD by 7.39 ms. In individual sports, the differences were statistically significant. pvalue < 0.001. RRT for ISG showed a statistically superior and significant reaction of hand-D compared with the hand-nonD by 7.81 points, pvalue 0.025. RRT to visual stimuli revealed the following differences: between right hand-D compart with left hand-nonD 10.61 points; between left hand-D and right hand-nonD −6.57 points; between right-left hands-D 9.68 points; between right-left hands-nonD −7.50 points. RRT measured with Hit-the-dot test  had bettIn the TMT test , athletepvalue 0.001. About SRT, except for judo, wrestling had a more marked difference between D-nonD hands intersecting gymnastics, karate and judo. For individual sports at Hit-the-dot test, RRT differences between hand-D and hand-nonD were of 9.90 s, pvalue 0.001. In individual sports, CRT did not record interaction; all sports gave similar results; they were all on the same descending slope and all sports had recorded better times for hand-D versus hand-nonD. TMT-B for CRT, the differences between hands-D and hand-nonD were 0.82 s to individual sports, with no statistical significance. Interaction to TMT-B for CRT individual sports does not show significant differences, most of them have better times at hand-D. A significant difference between D-nonD hands is represented by gymnastics which intersects with wrestling and karate which intersects with judo.In the Benchmark test , the difpvalue 0.294. In individual sports, in the Benchmark test, left arm significantly performed better than the right arm, no matter which was dominant. In individual sports, in the Hit-the-dot test, the right hand significantly performed better than the left hand, no matter which was dominant, pvalue < 0.001. The group distribution of individual sports in terms of hand-D was homogeneous p2 0.035) differences between hands D-nonD were significant, but the effect size was small. In individual sports, only in the Hit-the-dot test (ηpvalue < 0.000. According to RRT, boxing had better times than taekwondo which had the lowest result, MD ± SD 6.67 ± 1.190, pvalue < 0.000. In CRT, karate had better time and taekwondo weaker time, MD ± SD 7.737 ± 3.10, but without a significant statistical difference. Our results are in accordance with previous studies. One study performed in 2017 on six volunteers who participated in the tests of simply reaction time found that intense sport results in increased reaction times and the light physical activity had a positive effect on human reaction time, resulting in a shorter reaction time in both simple and recognition tests [In SRT, boxing had a better time, taekwondo had a weaker time, MD ± SD 59.05 ± 7.633, on tests ,17,18,19on tests . Many ston tests ,23,24. Son tests . A studyon tests . The implementation of information technology in the sports field is increasingly complex, aiming at both monitoring the training process and evaluating the motor and functional parameters of athletes.For hand-D and hand-nonD, the better results of individual sports were for: simply reaction time—boxing; recognition reaction time—taekwondo; cognitive reaction—judo. In Individual sports for SRT, left arm significantly performed better than the right arm. In individual sports for RRT, right hand significantly performed better than the left hand. Regarding CRT in individuals, the right arm significantly performed better than the left hand, no matter which was dominant. No significant differences were found between right or left hands in the TMT test.The more complex the application is, the more efficient the dominant hand is. Considering the simple reaction time, recognition and cognition through test computer games makes it possible to get an easy and attractive sports assessment. Future research will extend the research line by applying our results to other sports, on different sport age categories by analyzing the differences between D-nonD hands through other types of tests of computer games.


The model peptide was characterized by the highest ionization efficiency of all the proposed model podocin tryptic peptides in a canine urine sediment according to the LC-MS/MS analysis. The obtained results revealed the presence of the model peptide in 40.9% of dogs with MMVD  and 33.3% dogs with chronic kidney disease. The potential applicability of the developed methodology in the analysis of podocin in canine urine sediments was confirmed.The early asymptomatic stage of glomerular injury is a diagnostic challenge in the course of renal and extra-renal disease, e.g., heart insufficiency. It was found that podocin, a podocyte-specific protein present in the urine, may serve as a biomarker in the diagnosis of glomerular disease in humans and animals including glomerulonephritis, glomerulosclerosis, amyloidosis, or nephropathy. Therefore, there is a need of development of the sensitive and straightforward method of urinary podocin identification. In this work, we report our extended research under the glomerular injury investigation in dogs by application of clinical examination and LC-MS-MRM method in the identification of canine podocin in urine samples. The LC-MS-MRM method is based on the identification of podocin tryptic peptide with the  Podocytes are glomerular epithelial cells that line the urinary side of the capillary loops. This highly specialized cells completely enwrap the glomerular capillaries with their primary and secondary processes. These processes combine with the neighboring foot processes forming a so-called slit diaphragm. Because of their anatomic localization at the outer aspect of the capillary loops, podocytes are constantly challenged not only by oxygen radicals, cytokines, immune complexes, and inflammatory processes but also by mechanical forces  ,2,3,4. NUrinary podocytes are diagnosed based on the presence of associated proteins, such as podocin, nephrin, podocalyxin, synaptopodin, Wilms tumor protein-1, glomerular epithelial protein-1, and alpha actinin-However, to date, a podocyte-specific marker has not been identified. Podocin is one of the most robust biomarkers of podocytes . It may 39H-QEAGPEPSGSGR-OH50 sequence was chosen as most promising for podocin identification and quantification in urine sediments, and the obtained results were in agreement with the immunological tests. Additionally, in 2014, the new approach for podocin quantification in the soluble fraction of urine, instead of urine sediments, were presented by Simon and co-workers 2+ (m/z 748.0) ion, as the most intensive signal on the obtained mass spectra. The following transitions 748.0→143.0 (b2), 748.0→997.5 (y10), 748.0→754.5 (y7), 748.0→1110.6 (y11), 748.0→272.1 (b3) were selected after manual development of the MRM method and used in the investigation of podocin in the canine urine sediment samples. The retention time of the chosen peptide under chromatographic separation conditions presented in materials and methods section was 3.8 min. The samples were prepared according to the procedure described in the materials and methods section. The obtained results for the sample obtained from the animals with diagnosed MMVD  and CKD are presented in To choose the best peptide for canine podocin identification that may be present in trace amount in urine samples, the sensitivity of detection for all of the obtained compounds was analyzed using LC-MS/MS and LC-MS-MRM methods. The obtained data clearly confirmed that peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence with the confirmed retention time. Based on the obtained MRM chromatograms, it can be assumed that podocin was found in the analyzed samples. The obtained MRM chromatograms for the analyzed urine sediment samples originating from animal with diagnosed MMVD  and CKD The data obtained in the clinical and additional examinations for the analyzed dogs are presented in Dogs in the heart and kidney groups were, statistically, significantly older than the healthy dogs. They have also significantly higher SAP . There wAll the dogs in the control group had normal values of urea, creatinine, Cyst C, and SDMA. The kidney group dogs with abnormal high urea and creatinine level had elevated, SDMA, Cyst C, and AspAT concentrations compared with control group. The heart group dogs had statistically lower iron concentrations in blood serum and lower Cyst C level than the healthy dogs. The levels of creatinine, SDMA, and chloride were statistically higher in the kidney group than in the heart group. Despite the average, level of aldosterone was visibly higher in heart and kidney groups vs. control; the large individual variations made the difference statistically not significant .The urine specific gravity as well as blood urine and UAC were significantly lower in the heart and kidney groups compared to the control group. Despite the average, level of detection of podocytes using LC-MS-MRM method was visibly higher in heart and kidney groups vs. control; the large individual variations made the difference statistically not significant . LA/Ao aSpearman’s correlation indicated that the increase in urine creatinine values strongly correlated with an increase in urinary podocytes in the control and kidney group, while no such association was observed in the heart group . In the Here, we present our investigation on the application of LC-MS-MRM method in the analysis of podocin tryptic peptides in canine urine samples as a potent podocyte biomarker and correlation of the obtained results with the clinical, hematological, and biochemical data and urine test results.g, centrifugation is more useful for the diagnosis of podocin than the urinary supernatant. The low speed preserved the podocyte damage (the cell membranes) and the release of podocin into the supernatant. Due to the fact that there are no commercial tests dedicated to the detection of canine podocin, it was necessary to first establish the best biological material for the study by testing urinary sediment and supernatant . We foun79H-LQTLEIPFHEVVTK-OH92, 123H-AIQFLMQTTMK-OH133 150, H-SIAQDLK-OH156, 218H-AAEILAATPAAVQLR-OH232 were chosen, which may influence the ionization efficiency during LC-MS-MRM analysis. Finally, we focused our attention on a tryptic peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence because it is located outside the transmembrane region and gives intensive signals during the MS/MS experiment corresponding to the b2, b3, y7, y10 and y11 fragment ions. Moreover the 218H-AAEILAATPAAVQLR-OH232 sequence meets the peptide selection criteria described by Mohammed and co-workers pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) and trifluoroacetic acid (TFA) were obtained from IrisBiotech. Solvents for peptide synthesis , dichloromethane (DCM), and (N-ethyldiisopropylamine (DIEA) and tetraethylammonium bicarbonate (TEAB) were obtained from Sigma Aldrich; triisopropylsilane (TIS) was purchased from Fluka . Amicon® Ultra Centrifugal Filters were purchased from Merck .All the solvents and reagents were used as supplied. Fmoc amino acid derivatives and the Fmoc-Arg(Pbf)-Wang resin (0.32 mmol/g), Fmoc-Lys(Mtt)-Wang resin (0.56 mmol/g) were purchased from Novabiochem. Model peptides on the Fmoc-Lys(Mtt)-Wang and Fmoc-Arg(Pbf)-Wang were synthesized manually in polypropylene syringe reactors (Intavis AG) equipped with polyethylene filters, according to a standard Fmoc (9-fluorenylmethoxycarbonyl) solid phase synthesis procedure.2O, 4:1) over 30 min (flow rate 1 mL/min). The main fraction, corresponding to the peptide, was collected, lyophilized, and confirmed by MS/MS experiment.The synthesized peptide was purified using the analytical HPLC Thermo Separation system and UV detection (210 nm) with a YMC-Pack RP C18 column , and a gradient elution of 0–40% B in A  equipped with standard ESI source. The instruments were used in the positive-ion mode and calibrated with the Tunemix™ mixture . The mass accuracy was higher than 5 ppm. The analyte solutions (70 μL) were introduced at a flow rate of 3 μL/min. The instrument parameters were as follows: The scan range of the micrOTOF-Q MS was 50–1600 +) and doubly protonated ([M + 2H]2+) precursor ions were selected on the quadrupole and subsequently fragmented in the hexapole collision cell. Argon was used as the collision gas. The obtained fragments were registered as an MS/MS (tandem mass spectrometry) spectrum. The collision energy (10–30 V) was optimized for the best fragmentation. Bruker Compass DataAnalysis 4.0 software was used for MS spectra analysis.The singly . In the next step, 100 µL of 0.2 M DTT was added, the sample was incubated for 1 h at 30 °C and centrifuged at 4000 rpm for 15 min. DTT was removed by washing the sample with a 0.1 M TEAB buffer and centrifugation. The groups were blocked by the reaction with 100 µL of 0.1 M CAM . Excess of CAM was removed by washing the sample with 0.1 M TEAB and by centrifugation. The obtained supernatant was placed into an Eppendorf tube. Then, 50 μg of trypsin was added in 200 μL of 0.1 M TEAB, and the sample was incubated at 37 °C overnight. After digestion, 20 μL of formic acid was added, and the sample was lyophilized. The dry solid was dissolved in 5% MeCN/H2O and used for the LC-MS analysis. The urine sediment was stored up to a month at −80 °C and was thawed prior to analysis. After thawing, podocin was detected according to the following protocol. The collected urine sediment samples (0.7 mL each) were centrifuged for 10 min at 5000 rpm at 30 °C. The supernatant was discarded, and the pellet was re-suspended in 1 mL of 0.1 M TEAB buffer containing RapiGest™ SF at a concentration of 0.1% and sonicated for 5 min. The sample was transferred into the Amicon79H-LQTLEIPFHEVVTK-OH92, 123H-AIQFLMQTTMK-OH133, 150H-SIAQDLK-OH156, 218H-AAEILAATPAAVQLR-OH232 sequences were chosen as a potential podocin biomarkers in canine urine sediments. The peptides containing C-terminal arginine or lysine residue which may influence the ionization efficiency during LC-MS-MRM analysis. Finally, we focused our attention on a tryptic peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence  were from 5 to 80% B within 13 min. The flow rate was 0.3 mL/min, and the injection volume was 5 μL. The MRM method was developed manually, and the following transitions were chosen: 748.0→143.0 (b2), 748.0→997.5 (y10), 748.0→754.5 (y7), 748.0→1110.6 (y11), 748.0→272.1 (b3).LC-MRM experiments were performed on an Agilent Technologies 6470 Triple Quad LC/MS apparatus, with an Agilent Technologies 1290 Infinity II system equipped with an Aeris Peptide XB-C18 column (50 mm × 2.1 mm) 3.6 μm bead diameter, equilibrated at 24 °C. The LC system was operated with a mobile phase, consisting of solvent A: 0.1% formic acid in Hp value < 0.05 was considered significant. Statistical analyses were performed using StatSoft Statistica PL 12.0 Software. Data were expressed as mean and standard deviation (±SD) or median (dependent of distribution of variables). The normal distribution of data was analyzed using the Shapiro-Wilk test. One-way ANOVA with the Fisher’s LSD post-hoc test was used to compare normally distributed variables. The Kruskal-Wallis test was used for non-normally distributed or non-parametric variables. A simple or multiple ordinal regression test was used to describe the relationships between variables. A The research does not require the consent of the ethics committee because the experimental procedures did not go beyond the framework of clinical diagnostics.218H-AAEILAATPAAVQLR-OH232 sequence using LC-MS-MRM method. The presented strategy allows us to identify the presence of podocin in canine urine. We found that urinary sediment is more useful for the diagnosis of podocin than the urinary supernatant.We demonstrated a new methodology for the detection of podocytes in canine urine samples based on the identification of a podocin tryptic peptide with the LC-MS-MRM method is an exceedingly sensitive method, allowing the distinction of active kidney injury as it detects podocin in animals with glomerular injury (CRS and MMVD). Diagnostics of podocyturia using LC-MS-MRM method is a very promising method, which can greatly facilitate the unambiguous identification of podocin, could improve the diagnostic of early stage of glomerular injury, distinguish the active stage from inactive, and facilitate monitoring of patients’ treatment to avoid kidney damage during therapy. LC-MS-MRM method confirmed podocyturia in 40.9% of dogs with MMVD and 33.3% dogs with CKD. In accordance with our assumptions, podocin was not found in the urine samples of healthy dogs. The limitation of our study was the detection of a single podocin marker and a relatively small number of dogs. In dogs, this marker does not differentiate podocytes, PEC, and PTEC. On the other hand, all these cells are present in active glomerulonephritis and should not be detected in healthy animals. It is likely that different podocyte populations (depending on the dominant protein) were diagnosed at various stages of the disease. Hence, the use of a selection of different proteins targeting podocytes would increase the sensitivity of the method. Podocyte-specific proteins should be located in different cell compartments: In the glycocalyx, the cytoplasm, and on the basal side of the podocytes.


Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis. MicroRNAs of the miR-371-373 cluster, which are produced in embryonal tumors, have been proposed as sensitive biomarkers for testicular germ cell tumors (GCT), both in primary cases and recurrences . miR-371Detection of specific, free, circulating microRNAs in serum and plasma as biomarkers is warranted as it is a simple, non-invasive technique with little discomfort for the patient. In addition, medical staff, both in hospitals and primary health care, are trained to draw and handle blood samples. MicroRNAs have been shown to be stable in whole blood and separated serum and plasma for several hours . StandarThe minute amounts of specific microRNAs in serum and plasma need very sensitive methods for detection. Relative quantitation of microRNAs by reverse transcription real-time PCR (RT-qPCR) needs at least one reference gene in order to normalize samples, most preferably an endogenous microRNA (reference microRNA). Reference microRNAs must be expressed at similar levels throughout all samples tested, both in serum or plasma from individuals with the disease in question and in healthy controls. However, suitable reference microRNAs are often expressed at high levels in blood cells. Thus, the quantification is prone to error due to increased levels of the reference microRNA(s) caused by rupture of blood cells. Thus, hemolysis must be taken into account if the endogenous controls used are expressed in blood cells . Pre-anaHere we show how hemolysis affects four microRNAs used as reference genes in the quantitative analysis of miR-371a-3p and miR-372-3p. miR-93-5p, miR-20a-5p, and miR-30b-5p have been used as reference genes in analysis of the miR-371-373 cluster in serum . In addig for 10 min at 20°C within 1 h, aliquoted and frozen at –80°C. Absorbance at 414 nm was measured to assess baseline hemolysis in the serum samples. Red blood cells (RBCs) were collected from a healthy donor by centrifugation of 3 mL K2EDTA whole blood, with removal of plasma and buffy coat. The RBC fraction was hemolyzed by sonication. Serum was collected from the same individual and processed as described for the patient samples for use in the preparation of hemolysis standard curves described below.In this study, we analyzed serum samples from two patients diagnosed with testicular germ cell cancer, Patient 1 and Patient 2. The blood was collected prior to orchiectomy in 10 mL BD Vacutainer vials with clot activator. After coagulation, serum was separated from the blood cells by centrifugation at 2000 × No, weak and medium hemolysis in aliquots of the patient’s serum samples were imitated by adding RBC lysate to 0.0% , 0.05% (Weak hemolysis) and 0.2% (Strong hemolysis) (v/v). Five replicates for each sample and hemolysis condition were performed.414) (Hemolysis was assessed by absorbance at 414 nm (A414)  in all t414 was recorded prior to RNA extraction from these serum samples and followed by RT-qPCR for miR-93-5p, miR-30b-5p, miR-20a-5p, and miR-191-5p as described for the patient samples, except omission of the pre-amplification step. The experiment was repeated trice. The raw Cq-values were used to visualize how the levels of the four microRNAs increased with increasing concentration of hemolyzed RBCs in serum.Standard curves for absorbance assessment of hemolyzed serum samples were made by serial dilutions of hemolyzed RBCs in serum from a healthy individual. The samples ranged from 0.0 to 1.0% (v/v) hemolysate. ATotalRNA including small RNAs was extracted from all samples using the miRNeasy kit . A standard volume of 200 μL serum was added to 1 mL Qiazol Lysis Reagent. A mix of synthetic spike-in UniSp4  and 3 μg glycogen  as carrier was added to the homogenate prior to phase-separation by chloroform. A standard volume of 500 μL upper phase was extracted and further processed as described by the manufacturer. RNA was eluted in 30 μL nuclease-free water.Template RNA, at a fixed amount of 2 μL in each reaction, was reverse transcribed into cDNA using the Universal cDNA Synthesis kit II . In order to monitor the cDNA synthesis step, the synthetic cel-miR-39-3p  was included in the reaction mix of the cDNA synthesis. The final reaction mix of 10 μL was then incubated in a thermocycler at 42°C for 60 min and 95°C for 5 min. Five cDNA replicas were made for each sample and each hemolysis condition. Due to the low levels of miR-371a-372 in serum, a pre-amplification step was performed for the patient samples . A primeThe microRNAs were quantified using the comparative Ct-method .-ΔΔCq.The calibrator sample was a patient sample with very low levels of miR-371a-3p and miR-372-3p. RQs are 2t-test with Levene’s F-test for equality of variances. P-values < 0.05 in the two tailed t-tests were considered significant.The study was approved by the Regional Ethics Committee of Central Norway . Patients included in the study gave written informed consent in accordance with the Declaration of Helsinki. All statistical tests were performed in IBM SPSS Statistics Version 25. Expression levels of the microRNAs were compared using the Student’s The serial dilution of hemolyzed RBCs in serum showed that the amounts of RBCs added ranged from a non-detectable to a strong red color by visual inspection . By eye,Quantification by RT-qPCR of the miRNAs proposed as endogenous controls in analysis of microRNAs 371a-372 in serum showed that the concentration of miR-191-5p and miR-30b-5p did increase in serum when hemolysis occurs, but the increase in miR-93-5p and miR-20a-5p were more pronounced . AccordiRaw Cq-values from RT-qPCR analysis of miR-30b-5p, miR-93-5p, miR-191-5p, miR-371a-3p, and miR-372-3p adjusted for variation in RNA extraction efficiencies according to UniSp4 are shown in P < 0.05). The differences in RQs between the “Weak” and “Strong”-hemolysis samples are also statistically significant lower (P < 0.05). When miR-30b-5p was used as endogenous control in these samples, the differences in RQs in “No hemolysis” versus “Weak hemolysis” were lower, but still statistically significant for miR-371a-3p in both Patient 1 and Patient 2  and for miR-372-3p in Patient 2 (P = 0.010), but not significant for Patient 1 (P = 0.112). miR-191-5p as reference microRNA gave miR-371a-5p and miR-372-5p RQs that were not statistically significant higher in the “No hemolysis” versus the “Weak hemolysis” samples .As described, two different patient samples were spiked with increasing amounts of hemolyzed RBCs. We measured the initial absorbance at 414 nm for the patient samples and again after spiking . The RBCP < 0.05). It is generally accepted that the mean of at least two reference genes should be used in relative quantification of microRNAs. We therefore calculated the RQs for miR-371a-3p and miR-372-3p with the average of the two best reference microRNAs, miR-30b-5p and miR-191-5p  and digital droplet PCR (ddPCR). Especially ddPCR holds great promise in analysis of rare targets with better precision and reproducibility, but equivalent sensitivity compared to RT-qPCR . DdPCR iTo conclude, hemolysis assessment is an important quality control when working with circulating microRNAs in serum and plasma. Visual inspection is not sufficiently sensitive for monitoring hemolysis. Further efforts for finding good reference genes and normalization strategies in RT-qPCR analysis of circulating microRNAs must go on until consensus is reached if these microRNAs are to be used quantitatively for diagnostic purposes and clinical decisions.The raw data used for calculations are available in Supplementary Data This study was carried out in accordance with the recommendations of Helse Vest HF’s guidelines and the Regional Ethics Committee of Central Norway with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Regional Ethics Committee of Central Norway.MM, BR, and OD contributed in conception, design, interpretation of results and drafting of the manuscript. MM and BR performed the laboratory work and statistical analyses. PG, BB, ÁK, and MB included patients. All authors contributed in manuscript revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epithelial-to-mesenchymal transition (EMT) has been implicated as an important step in the development of distant metastases. We therefore wished to study EMT status of primary breast carcinomas from patients who during follow-up developed distant metastases.n = 151) from patients who developed metastatic disease were analyzed and EMT status was designated using a previously described EMT-core signature. EMT status of the primary tumor was correlated to clinicopathological characteristics, molecular subtypes, metastasis pattern, chemotherapy response and survival outcomes. In addition, using immunohistochemistry, the expression levels of several proteins implicated in EMT were studied  compared with the designated EMT status and survival.mRNA expression profiles of primary breast carcinoma samples (p < 0.001). EMT status was not correlated to the pattern of metastatic disease, metastasis-specific survival, and overall survival. Similarly, there was not a significant association between EMT status of the primary tumor and chemotherapy response in the metastatic setting. Immunostaining for NAT1 and TWIST1 correlated with the EMT status . Multivariate analyses showed that NAT1 and TWIST1 staining was significantly associated with EMT status regardless of the estrogen receptor status of the tumors .Utilizing the 130-gene-EMT-core signature, 66.2% of the primary tumors in the current study was assessed as EMT-activated. In contrast to our expectations, analyses revealed that 84.6% of Luminal A tumors, 65.1% of Luminal B tumors, and 55.6% of HER2-like had an activated EMT status, compared to only 25% of the basal-type tumors  contains supplementary material, which is available to authorized users. Snail, Twist, Slug, and Zeb1 and is regulated by the modulation of multiple epigenetic regulatory mechanisms  have been reported previously . Full inThe generated data were analyzed with help of R2 . Molecular subtypes were assessed for each tumor using the Pam50 classifier . Also thz scores were calculated for each tumor to define the EMT status in accordance with distribution of the z scores. EMT status identified by K-means method and z-scores of the tumors were subsequently compared to verify the identified EMT status of the given tumor. Given the significant concordance in assigned EMT status with K-means method and the z scores method (p 3.92e-20) in our dataset, further analyses and comparisons were carried out on the EMT status based on K-means method.To designate the EMT status of each tumor, the EMT-core gene list of Groger et al. was utilized . The 130n = 23 and not-EMT-activated, n = 23) were selected. The heat map created by supervised clustering with the EMT-core signature was carefully observed. Based on the current literature information on their established role in EMT, a subgroup of 7 proteins  was selected for further evaluation. To test the representativeness of this subset of genes, respectively, a K-means clustering method and a t test were carried out first to classify the tumors into two groups according to their EMT status and then to validate the performance of this classification in the same study set .To further reconcile the EMT-ness with the help of immunohistochemical stains, a subset of 46 tumors  were selected for the additional immunophenotypic evaluations.Whole-mount slides of the tumors from a subgroup of patients , N-cadherin , NAT1 (Abcam), SNAI2 (Abcam), Vimentin , and ZEB1 (Sigma Life science) was performed using an automated slide preparation system . The signal detection for immunohistochemistry was performed with a biotin-free ultraview universal DAB detection Kit . Immunohistochemical staining for TWIST1  was performed manually with a Bright DAB detection.Immunohistochemical staining for NAT1, SNAI2, TWIST, and ZEB1, a cut-off value of 10% was used to designate tumors as positive or negative. Absence of E-Cadherin expression in > 1% of the tumor cells was considered as loss of expression and presence of N-cadherin and Vimentin expression in > 1% of tumor cells was noted as gain of expression. Immunohistochemical findings were then correlated to the designated EMT status.The immunostained slides were scored by two pathologists . The invasive edges and the main tumor mass were evaluated separately. Immunostaining patterns for the other antibodies were evaluated semi-quantitatively; the extent and the intensity of the expression were scored for each antibody. For versus negative)] were utilized for the comparisons of the immunostaining results to the EMT status. Hazard ratios (HRs) were presented with their 95% confidence intervals (CIs). The statistical tests were two-sided and p value being less than 0.05 was considered to be significant.The association between the EMT status of the tumors and clinical variables was further investigated with multivariate logistic regression tests applying SPSS Statistics for Windows . Univariate and additional multivariate regression tests [adjusted for ER  of the primary tumors in the current study was assigned as EMT-activated and 33.8% (n = 51) as not-EMT-activated. The heat map shown in Fig. n = 274) of the tumors was identified as EMT-activated and 27.1% (n = 102) as not-EMT-activated.Using the 130-gene-EMT-core signature, 66.2% (p < 0.001). Histologic type or the size of the tumor was not found to be correlated with the gene expression-based EMT status (p 0.635).Comparisons of the histopathological characteristics of the tumors from EMT-activated group with the tumors from not-EMT-activated group showed that 76.9% of Grade 1 and 78.5% of grade 2 tumors had an activated EMT status as opposed to the 45.5% of grade 3 tumors (p < 0.001). Of Luminal A tumors 84.6%, of Luminal B tumors 65.1%, and of HER2-like 55.6% was assessed as having an activated EMT status, whereas only 25% of the basal-type tumors were assigned to the EMT-activated group (p < 0.001). Similarly, in the independent dataset [p < 0.001). Mean tumor percentage in the basal-type tumors was 72.1% (range 66.8–77.3). In the non-basal tumor groups, mean percentage was 58.9% in luminal A type, 65.8% in luminal B type, and 62.1% in HER2-like tumors (p < 0.001).Among ER-positive and PR-positive tumors 77.8% and 81.1% were identified as EMT-activated, respectively . Regarding the metastasis site, out of 108 patients who developed bone metastasis 72.2% had an EMT-activated primary tumor (p 0.021) versus 65.2% of the tumors of the patients with visceral metastasis (p 0.698).EMT status did not differ between the patients who developed metastasis within 5 years’ time and the ones who developed metastatic disease later than 5 years (p 0.162). Metastasis-specific survival time was 33 months for the patients with EMT-activated tumors and 19 months for those with non-EMT-activated tumors (p 0.036) .118 patients underwent chemotherapy treatment; 48.1% of the patients with EMT-activated tumors showed a response versus 31.4% of those with not-EMT-activated ones ; the findings are summarized in Table 2.Immunohistochemical evaluation for all stains revealed comparable expression patterns at the invasive edges and at the center of the tumors; therefore, analyses were further carried out based on a single score.E-cadherin, N-cadherin, Vimentin, SNAI2, and ZEB1 did not differ between the EMT-activated and not-EMT-activated groups .The expression patterns of staining for NAT1 expression was scored as positive in 25 cases with a range of 30–100% positivity in the tumor cells. Out of positively stained cases, 72% was assigned to the EMT-activated group; of negatively stained cases 76.2% was marked as not-EMT-activated (p 0.003). Further evaluations showed that NAT1 expression was not significantly correlated to the overall survival (p 0.223) and metastasis-specific survival (p 0.146).TWIST1 expression was found to be positive in 13 cases with 76.9% of these tumors being EMT-activated and of 60.6% of TWIST1 negative tumors belonged to the not-EMT-activated group (p 0.047). TWIST1 expression was not found to be significantly correlated with overall survival and metastatic-specific survival . Figure NAT1 and TWIST1 staining to predict EMT status, multivariate regression analyses were applied. Multivariate analyses results are displayed in Table NAT1 and TWIST1 staining was significantly correlated to EMT-activated status independent of ER status of the tumor .To investigate the additional role of In this study, gene expression profiles from primary breast carcinomas of patients with known metastatic disease have been utilized to assess the EMT status of the primary tumor. Subsequently, the designated EMT status has been correlated to the metastatic behavior and survival outcomes. In addition, the expression of EMT-associated proteins to the EMT status as assessed by gene expression profiling was studied.The previously suggested reciprocal link between basal-type breast cancer as assessed by immunohistochemistry and EMT  was not Mes) profile appeared to have better prognosis than the ones with epithelial (Epi) profile. The authors have suggested the role of stromal component and the distribution of molecular subtypes for the contradictory results. Concordantly, in this study, we have demonstrated that the percentage of tumor cells, hence the epithelial component differed among the molecular subtypes and luminal type tumors, had relatively more stromal component than the basal-type tumors, which may explain the association between luminal type tumors and the EMT-core signature (p < 0.001).The role of epithelial-to-mesenchymal transition (EMT) in cancer progression has been demonstrated in several tumor models. Yet, the translation of this concept to clinical breast cancer remains problematic and it has been argued that EMT may not be required for the development of distant metastases . Recent Next to its association with cancer progression and metastasis formation, EMT has been linked to chemoresistance in several cancer types , 62, 64.CDH1, CDH2, NAT1, SNAI2, TWIST1, VIM, and ZEB1. We were not able demonstrate the significant difference between EMT-activated and not-EMT-activated group regarding CDH1, CDH2, SNAI2, VIM, and ZEB1 expression, in the tumor bulk as well as at the invasive edges of the tumor tissue. Noteworthy, the staining pattern of TWIST1 and NAT1 have appeared to be related to the EMT status of the primary tumor. We have already pointed out the link between NAT1 and EMT status in a previous gene expression profiling-based study [NAT1) is a human enzyme that metabolizes arylamine and hydrazine-class drugs [NAT1 has been proposed as a useful biomarker for breast cancer [NAT1 led to the modification of intracellular actin which resulted in suppression of invasion and metastasis. Loss of NAT1 has also been reported to reduce the cell-to-cell contact growth and enhance the up-regulation of E-cadherin [Activation of an EMT program has been suggested as a critical event for cancer progression which grants epithelial cancer cells with more invasive mesenchymal phenotypes . Direct ed study  and its ed study , 42, 49.ss drugs . Next tot cancer , 50, 51.cadherin , 57, 63.cadherin , 58. Thecadherin , 47, 61.Many investigators have faced difficulties to detect cancer cells with EMT phenotype. To overcome the main obstacle which is to differentiate the stromal fibroblasts from the cells with EMT phenotype, Yu et al. conducted a study using RNA in situ hybridizations on HER2-positive breast tumors in order to distinguish primary tumor cells from the surrounding stromal cells . By usinNAT1 and TWIST1 may be of help to identify the tumor cells with EMT phenotype. We believe that our study is a valuable addition to the current literature and gives additional perspective on EMT in human metastatic breast carcinomas.In conclusion, our results fails to draw a direct line between the gene expression-based EMT status of a primary tumor and its associated metastatic behavior. Our findings suggest that the EMT status of the tumor, defined by the EMT-core signature, may largely be the result of the amount of stroma in the tumor (which is often larger in grade 1 tumors compared to grade 3 tumors). In this study, we have also demonstrated that immunostaining for Below is the link to the electronic supplementary material.Supplementary material 1 (XLSX 170462 KB)Supplementary material 2 (DOCX 69 KB)


Further, the preparation of poly(methyl methacrylate) and polystyrene nanoparticles (NPs) by mini-emulsion polymerization using AUPDS is investigated, leading to NPs with active ester groups on their surface. By systematically varying reaction parameters and reagent concentrations, it was found that AUPDS feed concentrations between 2–4 mol% yielded narrowly distributed and stable spherical particles with average sizes between 83 and 134 nm for non-cross-linked NPs, and up to 163 nm for cross-linked NPs. By basic hydrolysis of the active ester groups in aqueous dispersion, the positive ζ-potential (ZP) was converted into a negative ZP and charge quantities determined by polyelectrolyte titrations before and after hydrolysis were in the same range, indicating that the active ester groups were indeed accessible in aqueous suspension. Increasing cross-linker amounts over 10 mol% also led to a decrease of ZP of NPs, probably due to internalization of the AUPDS during polymerization. In conclusion, by using optimized reaction conditions, it is possible to prepare active ester functionalized NPs in one stage using AUPDS as a surfmer in mini-emulsion polymerization.Functional surface active monomers (surfmers) are molecules that combine the functionalities of surface activity, polymerizability, and reactive groups. This study presents an improved pathway for the synthesis of the active ester containing surfmer  Polymeric nanoparticles (NPs) may exhibit unique physical properties that differ from the properties of their counterparts in the macroscale. The differences are mainly based on the smaller object size, leading to a higher surface-area-to-volume ratio, a better dispersion stability, a larger diffusibility, and mobility. This dispersion allows the NPs to be generally more interactive with the external media than micro- and macroparticles ,2,3,4. Preparation of polymeric NPs with a narrow distribution allows for reliable and reproducible results for their applications, and heterophase polymerization of  water-insoluble monomers through emulsion polymerization (EP) and mini-emulsion polymerization (miniEP) are widely-employed methods to achieve monodisperse size distributions. Surfactant-based approaches may be used in both EP and miniEP systems in order to prevent the coalescence of monomer droplets via electrostatic and/or steric stabilization. In the case for EP, micellar nucleation is prevalent and the surfactants are usually used above the critical micelle concentration (CMC) ,6. In coIn order to make use of the large surface-area-to-volume ratio of polymer NPs, the NP surface is often decorated with groups which bear a chemical, physical or biological function. Such NPs can be regarded as functional NPs. Developing efficient ways to synthesize functional polymer NPs has been a notable field of study in chemistry and biotechnology ,12,13,14The most common approach for the generation of NP surfaces presenting defined functional groups is chemical modification after NP preparation. However, this post-polymerization approach often relies on multi-step reactions and a need for rigorous and time-consuming purification ,12. ThesDuring polymerization, the surfmers are directly incorporated into the polymeric backbone of the particles—while predominantly present on the particle surface. Hence, the use of functional surfmers allows for a controlled display of the functional head groups with a covalent attachment to the surface of the particles ,24,27,28p-(11-acrylamido)undecanoyloxyphenyl dimethylsulfonium methyl sulfate (AUPDS), a water soluble surfmer that presents an active ester functionality that acts as an anchor group. AUPDS was synthesized by Herold et al., who used it in the preparation of polymer NPs, but only through the EP technique [As aforementioned, a wide range of surfmer molecules has been developed so far and polymeric particles have been developed using surfmers bearing cationic ,30,31 orechnique . The actechnique ,44. Surfechnique  and surfechnique ,47. Using surfmer molecules to obtain functional particles systems that are both monodisperse and reproducible is a challenging task and, for a better understanding of the mechanistic approach of miniEP using surfmers and of the colloidal properties of the obtained particles, a systematic study of different formulations is necessary. The aim of this work is to investigate the role of AUPDS as a surfactant and co-monomer in a thermally-initiated miniEP system using either styrene (St) or methyl methacrylate (MMA) as co-monomers. Cross-linked particles and the effect of varying cross-linker concentrations in these surfmer-functionalized particle systems were also investigated. The obtained particle properties, such as particle size, distribution, and surface charge were considered and the different formulations were compared.N,N′-diisopropylcarbodiimide 99% (DIC), potassium dihydrogenphosphate, potassium hydroxide, sodium hydrogen phosphate, sodium hydroxide, and styrene (St) ≥ 99% were obtained from Sigma-Aldrich . From other suppliers: 11-Aminoundecanoic acid 97% ; ethyl acetate ; deuterated dimethyl sulfoxide (DMSO-d6) 99.8% Deutero, Kastellaun, Germany); hexadecane 98% ; (4-hydroxyphenyl)(dimethyl) sulfonium methyl sulfate ; and sodium polystyrene sulfonate ; poly . Monomers were distilled under reduced pressure and stored under argon (Ar) at −20 °C. All solvents used were of HPLC grade or higher. Phosphate buffers of pH 7.0 and 7.5 were prepared using potassium dihydrogenphosphate and sodium hydrogen phosphate solutions. Ultrapure water  was used to prepare all aqueous solutions and is hereby referred to as water. The following reagents were purchased from commercial suppliers and, unless stated otherwise, were used as received. Acetonitrile, acryloyl chloride 97%, azobisisobutyronitrile 98% (AIBN), chloroform, ethylene glycol dimethacrylate (EGDMA), dichloromethane (DCM); 4-(dimethylamino)pyridine 99% (DMAP), divinylbenzene (DVB), hydrochloric acid 37%, methyl methacrylate (MMA) 99%, 1H-NMR) spectroscopy was performed using an AVANCE 500 spectrometer , standardized to 2.50 ppm with DMSO-d6. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra were measured using an Equinox-55 spectrometer  equipped with a deuterated triglycine sulfate (DTGS) detector and the corresponding software OPUS.Proton nuclear magnetic resonance (−1) in a 4-necked 1 L flask, equipped with mechanical stirrer, argon (Ar) inlet, thermometer, and dropping funnel. Acryloyl chloride  was dissolved in 15 mL DCM and slowly added dropwise into the reaction under Ar atmosphere and ice bath. After the addition was completed, the reaction was left stirring at room temperature (RT) for 16 h. The reaction was adjusted to pH 7.0 via dropwise addition of HCl (1 mol·L−1). The colorless and opaque reaction mixture was then filtrated and the solid filtrate was washed five times with HCl solution (0.1 mol·L−1), followed by five washes with water. The resulting white powder was dried under vacuum overnight and was used for the next step without further purification (crude yield: 78%).11-Aminoundecanoic acid  was dissolved in 150 mL of aqueous KOH solution (1.3 mol·L1H-NMR and ATR-FTIR spectra are available in the The second step of the synthesis consists in the Steglich esterification of the obtained 11-acrylamidoundecanoic acid (AAUA). HPDMSMS  was dissolved in 170 mL of acetonitrile and added to a solution of 11-acrylamidoundecanoic acid  in 370 mL of chloroform under stirring and Ar atmosphere. A solution of DMAP  in 5 mL acetonitrile was added dropwise, followed by dropwise addition of a solution of DIC , in 15 mL of acetonitrile, under ice bath. The reaction proceeded under stirring at RT for 72 h. A white precipitate was removed through filtration and the volatile solvents of the filtrate were evaporated, resulting in a white powder. 50 mL of ethyl acetate were added to the powder and the mixture was left stirring at room temperature for 15 min, then the solids were filtered. This process was repeated three times in order to remove the DIC by-product. The white powder was then dried under vacuum (yield: 89%). 1H-NMR (DMSO-d6): δ (ppm) = 8.17–8.10 , 8.07 , 7.50 , 6.25–6.16 , 6.05 , 5.55 , 3.38 , 3.27 , 3.11 , 2.62 , 1.64 , 1.46–1.20 .ATR-FTIR: 3308 ; 3018 ; 1587 and 1475 ; 2918 and 2849 ; 1759 ; 1653 ; 1626 ; 1533 .For the synthesis of polymeric NPs via mini-emulsion polymerization, a continuous phase (water) was mixed with a dispersed (monomer) phase, which consisted of monomer (either St or MMA), AIBN (initiator), HD (hydrophobe), and AUPDS surfmer, which was used as the only surface active compound. Throughout the formulations, AUPDS surfmer content varied from 0.25 to 8 mol%, AIBN and HD were fixed at 0.5 and 2 mol%, respectively, and cross-linkers (when present) varied from 0.5 to 80 mol%. The term mol% refers only to the compounds on the dispersed phase. Examples of amounts of the dispersed phase for non-cross-linked formulations with AUPDS 2 mol%: 0.94 mg MMA (9.39 mmol), 77 mg AUPDS (0.20 mmol), 8 mg AIBN (0.05 mmol) and 45 mg HD (0.20 mmol); or 0.91 g St (8.74 mmol), 72 mg AUPDS (0.18 mmol), 8 mg AIBN (0.05 mmol), and 41 mg HD (0.18 mmol). AUPDS was dissolved in the water (continuous phase), which was maintained at 6 mL (15–20 wt % of dispersed phase). Further examples of reagent amounts can be found in n = 3) were dried using a drying oven  at 60 °C and 125 mBar for 24 h. In order to achieve the described protocol, preliminary screening experiments were performed with non-crosslinked particles with 2 mol% surfmer and the aim was to find appropriate experimental conditions to generate mini-emulsions with the AUPDS surfmer. Reaction conditions, ultrasonication time, initiation and parameters for the centrifugation rounds were tested and the outcome was evaluated by observing visible changes  of the mini-emulsions, as well as by comparing the yield and particle sizes/dispersity after polymerization.The reagent mixture was then homogenized for 3–5 min at RT at approximately 600 rpm and, afterwards, it was mini-emulsified by ultrasonication using a probe sonicator  with 60% amplitude, 120 s of total sonication time (pulse set at 10 s on/5 s off) under an ice bath. The mini-emulsion flask was subsequently transferred to an oil-bath and the polymerization was carried out at 75 °C and 400 rpm for 4 h. After reaching RT, the particles were purified three times with water through centrifugation at 25,000 rpm, for 30 to 60 min at 10 °C, using an Avanti J-26 XPI centrifuge . Polystyrene (PS) particles were first washed once with methanol. After each centrifugation cycle, the precipitates were redispersed in water and the suspension was finally stored in the fridge. This stored suspension is hereby referred to as NP stock suspension. For determination of solid content of this stock suspension, aliquots of each suspension (The prepared particles were characterized through scanning electron microscopy (SEM) and dynamic light scattering (DLS), also referred to as photon correlation spectroscopy. The numerical size distribution is shown in terms of polydispersity index (PI) obtained via DLS. The presence of the surfmers on the surface of the particles was investigated through electrophoretic light scattering  and polyelectrolyte titration (for charge quantity), in which particles functionalized with AUPDS were used both unmodified and after a hydrolysis process. Hydrolysis of the active ester groups was accomplished by dispersing the particles in NaOH solution (0.1 M) overnight. Particles were recovered through centrifugation and were redispersed in sodium and potassium phosphate buffer .v/v), without prior use of ultrasonication or filtration processes. The measurements were executed and evaluated according to the ISO 22412 [DLS and ELS were performed in a Zetasizer Nano ZS device  using the corresponding software Zetasizer Nano ZS Version 7.12. Size measurements were executed in the non-invasive backscattering mode (NIBS) at a scattering angle of 173°, using PS disposable cuvettes. Zeta potential (ZP) was determined in ELS mode, using folded capillary zeta cells . All measurements were carried out in triplicate, at 25 °C, with the number of runs, attenuator value, and laser position set to automatic. Samples were prepared by further diluting the particle stock suspensions in water . Samples were prepared in the same manner as for DLS measurements and 5 μL droplets were placed on clean silicon wafers and left to dry overnight at room temperature. Polyelectrolyte titrations were carried out via potentiometry using a particle charge detector  coupled to a Titrino 702SM  using the corresponding BTG Mütek PCD software. All measurements were performed in triplicate, under agitation at RT, where 0.01 mL of titrant solution (c = 1 mM) was added to the colloidal suspension every 20 s, until the charge was neutralized. For sample preparation, at least 10 mg of dispersed particles were added to sodium and potassium phosphate buffer  until total volume of 15 mL. PES (anionic) was used to titrate positively-charged particles and PDADMAC (cationic) for the negatively-charged particles.1H-NMR and ATR-FTIR analysis confirmed the AUPDS structure and showed a conversion of 87% due to the presence of residuals from AAUA, which is comparable to the other procedure. Residual AAUA could not be reduced further due to the limited stability of AUPDS towards hydrolysis, excluding further extraction steps or column chromatography. Although the residual AAUA in the product can act as a surfactant itself, it is not expected to interfere with the particle synthesis described in the following sections as also supported by the report by Herold et al. [The AUPDS surfmer was synthesized by modifying a previous approach developed by Herold et al. . For thed et al. . Taken tThe choice of reagents and emulsification parameters play a decisive role in the preparation of particles through miniEP technique. The formation of mini-emulsion systems requires high mechanical mixing in order to reach a steady state, given by a rate equilibrium of droplet fission and fusion. The droplets are then critically stabilized and, during polymerization, the droplets act as “nanoreactors” because only droplet nucleation occurs . The genBecause the functional active ester group of AUPDS is present in the hydrophilic head group, we chose to perform direct mini-emulsion experiments with a hydrophobic monomer dispersed phase and water as the continuous phase. Thus, when using AUPDS as a surfactant in miniEP the active ester groups should be directed towards the water phase and should be present on the NP surface after polymerization. We chose the monomers MMA and St for particle synthesis because they are known to form monodisperse NPs in miniEP ,51. AddiThere are two factors to assure that nucleation only takes place in the monomer droplets: the type of the initiator and the surfactant concentration. In our formulations, the initiator of choice was AIBN, due to the fact that it is only soluble in the dispersed organic monomer phase. This is particularly important to prevent secondary nucleation in the continuous phase (water) for MMA which is partly soluble in water. The AUPDS molecule, due to its amphiphilic character, acts as a surfactant substitute while also acting as a co-monomer. By using low AUPDS concentrations below the critical micelle concentration (CMC), the surfmer does not cover the droplet interface completely ,53. ThisThe monomer droplets in mini-emulsion systems are stabilized against the Ostwald ripening effect via the use of co-stabilizers—the so-called (ultra)hydrophobes . Hexadecco-DVB and PMMA-co-EGDMA NPs (both cross-linked). The objective was to systematically test the effect of varying surfmer and cross-linker concentration in order to obtain monodisperse functionalized PMMA and PS NPs with reproducible properties and stability. In our experiments, we produced NPs with AUPDS as the only surface active agent: PS and PMMA NPs (both non-cross-linked) as well as PS-The AUPDS surfmer was used as surfactant replacement and co-monomer in the preparation of MMA and PS NPs with a surfmer feed concentration between 0.25 and 8 mol% relative to the dispersed monomer phase. Variations in the values of the hydrodynamic diameters, PI and ZP were used as parameters to evaluate particle size, distribution and stability and these results are displayed in When using formulations with low surfmer concentrations of 0.5 and 1 mol% AUPDS, PS particles presented an increased PI (0.16 and 0.37) and a low ZP (+8.7 to −4.5 mV), whereas PMMA particles were not even produced at this range. The reason for that is because this amount of surfmer was not enough to stabilize the mini-emulsion before polymerization. Our results are in agreement with the results obtained by Landfester et al. for SDS as the surfactant for the miniEP of St: when using less than 1 mol% of surfactant, there is just not enough coverage of the monomer droplets to stabilize the system . co-AUPDS particles with hydrodynamic diameters fluctuating between 118 nm and 150 nm and PI below 0.13. On the other hand, PS-co-AUPDS particles presented a slight decrease in the hydrodynamic diameter from 90 to 55 nm with increasing surfmer amounts ≥ 2 mol%, but size distributions were broader when using more than 4 mol% AUPDS (PI ≥ 0.23). An increase in the surfactant concentration usually leads to a decrease in the particle size due to the increase in the number of droplets in the mini-emulsion, provided that the droplets are covered with enough surfactant [co-AUPDS, but not for PMMA-co-AUPDS particles. After the minimum concentration is reached and a stable mini-emulsion is formed, it is possible to produce stable droplets with as little as 10–30% surface area coverage using miniEP systems . Variatirfactant . This waN-Alkyl acrylamides, for example, which we expect to have a similar co-polymerization reactivity like AUPDS, on average were reported to have a higher co-polymerization ratio with St than with MMA [co-AUPDS particles. The opposite could happen for St, where AUPDS is incorporated to a larger extent in the earlier stages of polymerization, thus decreasing the amount of molecules on the surface. It is also pertinent to emphasize that the solubility of the monomer (St or MMA) in water should not play a significant role in these systems because in the miniEP mechanism the nucleation takes place in monomer droplets, especially when using a hydrophobic initiator (AIBN), which particularly avoids the formation of water-soluble oligomers [In both mini-emulsions, the AUPDS acts as a stabilizer, replacing the use of surfactant, while also being a co-monomer to St or MMA. The inherent reactivity of the acryloyl polymerizable end group of AUPDS certainly has an influence on the surfmer distribution on the surface of the particles post polymerization, this could be mainly due to different reactivity ratios among the monomers . Ideallywith MMA ,58,59,60ligomers ,28.co-AUPDS and of PS-co-AUPDS using 2 mol% of surfmer are displayed in SEM micrographs of PMMA-co-AUPDS NPs varied with AUPDS concentration from 35 up to 43 mV. In contrast, the measured ZP values for PMMA-co-AUPDS NPs remained constant at about 44 mV with varying surfmer concentration. Considering the higher yield (at 88% for PMMA-co-AUPDS and 78% for PS-co-AUPDS), along with favorable DLS data, NPs were further synthesized using AUPDS at 2 mol% as the concentration of choice for both monomers. Among five batches using 2 mol% AUPDS, PMMA-co-AUPDS showed a Z-average size of 127.6 ± 14.8 nm, a PI of 0.05 ± 0.01 and a ZP of 42.2 ± 4.1 mV. Among four batches, PS-co-AUPDS particles had an average hydrodynamic diameter of 109.3 ± 26.4 nm, PI of 0.14 ± 0.05, and ZP of 33.9 ± 5.7 mV. The DLS measurements reporting the reproducibility of different batches for the optimized formulations are available on In regards to the ZP, PS-The preparation of cross-linked NPs is a common practice in order to provide a fixed structure that is resistant to different solvents systems and more thermally stable  althoughco-EGDMA-co-AUPDS promoted an increase in particle size (from 225 to 588 nm) and in PI from 0.29 to 0.40. The increase of EGDMA also led to a decrease in ZP values, from +15 mV to −25 mV. Therefore, it can be interpreted that an increase in the cross-linker amount from 20 mol% to up to 80 mol% leads to less AUPDS on the surface of the NPs. One of the reasons for this could be the fact that highly crosslinked PMMA-co-EGDMA are often porous particles [Increasing cross-linker concentration above 20 mol% did not yield stable mini-emulsions when using St/DVB and, for MMA/EGDMA the particles precipitated within days and, for that reason, higher ratios of cross-linker were not further explored. This increase in cross-linker content above 20 mol% for PMMA-articles , which wco-EGDMA-co-AUPDS particle sizes varied slightly from 168 to 159 nm and remained monodisperse with PI of 0.07 to 0.10, while the ZP varied from 31 to 52 mV. A similar effect was observed for PS-co-DVB-co-AUPDS particles, in which hydrodynamic diameters varied from 99 to 162 nm, PI remained monodisperse at 0.03 to 0.04, and ZP was in the same range at 26 mV. SEM micrographs of PMMA-co-EGDMA-co-AUPDS and of PS-co-DVB-co-AUPDS using 10 mol% of cross-linker are displayed in co-EGDMA-co-AUPDS had a Z-average size of 166.4 ± 3.1 nm, PI of 0.09 ± 0.01 and ZP of 41.1 ± 8.1 mV, whereas PS-co-DVB-co-AUPDS presented a Z-average size of 147.9 ± 9.7 nm, PI of 0.04 ± 0.01, and ZP of 29.2 ± 3.9 mV. All optimized formulations presented a satisfactory reproducibility with a low standard deviation.It was possible to produce stable and narrowly distributed NPs with both systems using a cross-linker range from 2.5 to 10 mol%. For PMMA-Coalescence of particles can be prevented by means of steric and/or electrostatic stabilization and the ZP is an indicative of the electrostatic particle stability in a suspension, as previously mentioned. These values can be positive or negative, depending on the molecular configuration in the surface of the particles, but it is not quantitative. A quantitative determination of the surface charge can be achieved via polyelectrolyte titrations. This approach allows for the determination of the effective dissociated counterion charge for particles and can be used for both positively- or negatively-charged particles . co-AUPDS, PS-co-AUPDS, PMMA-co-EGDMA-co-AUPDS, and PS-co-DVB-co-AUPDS particles using 2 mol% of AUPDS in the formulations, either non- or cross-linked with 10 mol% of cross-linker. In order to estimate whether the AUPDS surfmer was responsible for the surface charge and if it was available for chemical reaction on the surface of the particles we investigated the ZP and the charge quantity of the NPs before and after a basic hydrolysis process of the AUPDS active ester groups on the particles. We investigated this for PMMA-Due to the sulfonium group on the AUPDS, the particles should present a positive surface charge and, after hydrolysis, carboxylate groups should be present and the surface charge should be negative. As expected, the ZP value was positive for unmodified particles and negative for particles after hydrolysis, within the same value range. Moreover, the change in ZP for the NPs during basic hydrolysis also shows that the possible hydrolysis of PMMA side chains to their acid counterparts is not responsible for the ZP change  because co-EGDMA-co-AUPDS formulations remained in the same range as the non-cross-linked PMMA-co-AUPDS particles. On the other hand, the addition of cross-linker amplified the amount of surface charge from 9.9 μeq·g−1 of PS-co-AUPDS NPs to 48.0 μeq·g−1 of PS-co-DVB-co-AUPDS. This higher amount of surface charge is also consistent to the fact that these particles did not aggregate after the hydrolysis process and that aggregation after hydrolysis could have caused the smaller charge quantity found for PS-co-AUPDS particles. It is pertinent to emphasize that theZP values may also vary according to changes in the pH and in ionic strength of the dispersion media. The DLS and PCD measurements of The surface charge of cross-linked PMMA-co-AUPDS are the only formulation that significantly aggregated (three-fold increase in size and PI) and this could cause a hindering of the charged functional groups in the polyelectrolyte titrations. Altogether, these results show that the AUPDS active ester groups are indeed present at the NP surface after preparation and that they are accessible for chemical reactions in an aqueous NP suspension.From size measurements, the PS-Regarding the stability during storage, aqueous NP suspensions with a neutral pH stored at 4 °C maintained their ZP, size and PI for up to three weeks, while for suspensions stored at room temperature after 72 h the particle characteristics began to change. After this period, the ZP decreased and turned negative and the DLS and ZP data on stored particles can be found on co-EGDMA, PS and PS-co-DVB NPs copolymerized with the p-(11-acrylamido)undecanoyloxyphenyl dimethylsulfonium methyl sulfate (AUPDS) surfmer molecule via miniEP technique. The AUPDS surfmer was successfully synthesized and presented a high yield through a modified synthesis approach. The copolymerization of St or MMA with surfmer feed concentration of 2–4 mol% of surfmer yielded monodisperse and stable particles. Average hydrodynamic diameters, PI and ZP for the obtained particles were 127 nm, 0.05 and 42 mV for PMMA-co-AUPDS particles and 109 nm, 0.14 and 34 mV for PS-co-AUPDS particles when using 2 mol% AUPDS. For cross-linked particles, the results indicated that up to 10 mol% cross-linker yielded particles with low PI, with a slight increase in size. While maintaining the same AUPDS concentration ratios, increasing the cross-linker amounts also led to a decrease in the ZP of the obtained NPs, probably due to internalization of the AUPDS during polymerization. PMMA-co-EGDMA-co-AUPDS showed on average a hydrodynamic diameter of 166 nm, PI of 0.09 and ZP of 41 mV, whereas for PS-co-DVB-co-AUPDS the average was 148 nm, 0.04 and 29 mV. The presence of the surfmer on the NP surface was confirmed via ZP measurements and polyelectrolyte titration before (positive charge) and after a basic hydrolysis process (negative charge). In conclusion, using the optimized parameters functionalized NPs were produced in one step using the AUPDS surfmer. Furthermore, the active ester groups should allow to couple a wide range of amine-containing biomolecules to the particles which will be explored in future studies. The purpose of this work was an investigation of the preparation of PMMA, PMMA-


The genital system and skin involvement of diffuse B-cell lymphomas are quite rare. The appearance of these rare types in the same patient and the same period makes the treatment of the disease difficult. But both types respond well to anthracyclines and immunotherapies. A 74-year-old woman was treated with R-CHOP  without surgery and/or radiotherapy, and no recurrence at 2 years follow-up. Despite the poor prognosis of these types of lymphomas, treatment responses are quite good as they are in other subtypes. Malignant lymphoma accounts for 0.05% of uterine malignancies in women .We report the case of a woman who developed diffuse large B-cell lymphoma (DLBCL) on endometrium and on skin of lower extremity. Patient received six courses of R-CHOP and complete remission was achieved. It was observed that complete response was achieved for two fast-growing lymphomas.A 74-year-old postmenopausal female presented to dermatology outpatient clinic with the complaint of a mass on dorsum of the left foot and a mass on the middle part of the medial surface of the left lower extremity for a month. A tru-cut biopsy was obtained from malignant necrotic ecchymosis under antibiotic prophylaxis. Magnetic resonance imaging (MRI) of the pelvic and MRI of the lower extremity were performed. The results showed a mass obliterating the endometrium and a pathologic lymphadenopathy, measuring 30 × 15 mm, on the left inguinal region. Vaginal ultrasound was performed and adnexal atrophy and cysts in uterus were detected. Dilatation and curettage was performed on endometrium and patient was diagnosed with high-grade cancer. MRI of the lower extremity showed a heterogeneous mass, which is measuring 56 × 40 mm and is infiltrating proximal parts of third and fourth metatarsal bones and muscle and fat tissue on dorsal part of left foot. Positron emission tomography–computed tomography (PET-CT) was performed for stating. The results demonstrated a lesion, measuring 10.5 × 6.5 × 5.5 cm, extending from skin to extensor muscles and infiltrating proximal fourth and fifth phalanges in intermetatarsal spaces on plantar surface of the foot (SUVmax: 13.6). A primary lesion, measuring 25 mm in diameter, that is extending from medial calcaneus to subcutaneous tissue on proximal and one-third part of the middle part of left lower extremity (SUVmax: 26.5) and a second primary mass, measuring 45 × 28 mm, that is filling corpus of the uterus (SUVmax: 45.8) are noted. A metastatic lymph node, measuring 2 cm in diameter, is noted (SUVmax: 9.1) and2. Primary lymphoma of the endometrium is often detected by vaginal hemorrhage in elderly postmenopausal women without constitutional symptoms. Patients are treated with radical hysterectomy and lymph node dissection, followed by R-CHOP therapy. The 5-year overall survival rate is 39.3 %. In some cases, the complete remission rate of immunochemotherapy without surgery is 75%.Primary cutaneous B-cell lymphoma is often observed in elderly women. Lesions are present on head, neck, trunk, or lower extremity. Regardless of skin localization, MUM1/IRF4, bcl-2, and FOX-P1 are strongly expressed.i-67 in endometrial involvement were 60% positive.Two rare types of lymphoma detected in the same patient were treated with R-CHOP chemotherapy. A very good partial response was achieved at the end of three courses, and complete remission was achieved at the end of six courses. Considering that there was no residual disease and that sequelae effects of RT administered to the foot would adversely affect the quality of life, involved-field RT was not administered to the patient. In the literature, cutaneous or extracutaneous relapses have been reported frequently, despite good response to immunochemotherapy. In this case, there was no recurrence of both endometrium and skin for 2 years. Immunologically, B surface markers such as CD20, CD79a, Pax-5, and bcl-2 are frequently positive in diffuse cutaneous B-cell lymphomas.It is very rare to detect uterine corpus and cutaneous B-cell lymphoma, leg type, simultaneously in the same patient. During diagnosis, PET-CT and MRI examinations demonstrated involvements other than primary focus. Although both foci are fast growing histologically, it was observed that complete remission could be achieved with immunochemotherapy.


For the waitlist control group, significant reductions in plasma CXCL12 (SDF-1α) (p = 0.003), CXCL5 (RANTES) (p = 0.05) and BDNF (p = 0.003) were observed. A significant improvement in social connectedness was also observed in the HT group (p = 0.01). Conclusion: HT, in reducing plasma IL-6, may prevent inflammatory disorders and through maintaining plasma CXCL12 (SDF-1α), may maintain hematopoietic support to the brain. HT may be applied in communal gardening to enhance the well-being of older adults.The effect of horticultural therapy (HT) on immune and endocrine biomarkers remains largely unknown. We designed a waitlist-control randomized controlled trial to investigate the effectiveness of HT in improving mental well-being and modulating biomarker levels. A total of 59 older adults was recruited, with 29 randomly assigned to the HT intervention and 30 to the waitlist control group. The participants attended weekly intervention sessions for the first 3 months and monthly sessions for the subsequent 3 months. Biological and psychosocial data were collected. Biomarkers included IL-1β, IL-6, sgp-130, CXCL12/SDF-1α, CCL-5/RANTES, BDNF (brain-derived neurotrophic factor), hs-CRP, cortisol and DHEA (dehydroepiandrosterone). Psychosocial measures examined cognitive functions, depression, anxiety, psychological well-being, social connectedness and satisfaction with life. A significant reduction in plasma IL-6 level ( During the process of normal aging, there are neurobiological changes such as the loss of neurons , and psyA recent study by Li et al. 2010) reported increases in the activity of natural killer (NK) cells and its enzymes after a forest bathing trip . The aut reportedAlthough there have been numerous studies on HT, a systematic review of randomized controlled trials (RCTs) on HT concluded that the studies conducted so far were relatively low quality . In partIt has been postulated that increased levels of IL-6 and IL-1β are involved in the pathogenesis of Alzheimer’s disease and major depressive disorders . On the In view of this, Lin et al. suggested that measures of biomarkers should be incorporated in future studies . This prhttps://clinicaltrials.gov/ct2/show/NCT02495194). The participants were 59 older adults between the ages of 61 and 77  recruited from a neighbourhood in the western region of Singapore. All the participants were screened based on the inclusion and exclusion criteria. That is, the participants should be aged between 60 to 85 years old, able to provide informed consent and have a minimum Montreal Cognitive Assessment (MoCA) score of 22. They must not have current or past histories of severe medical or psychiatric disorders. They must not be undergoing any concurrent therapy, and should have no significant visual or hearing impairments or upper and lower limb motor difficulties. Subsequently, qualified participants were randomized into either the HT intervention group or waitlist control group.This trial began with the Ethics Board’s approval with NUS-IRB . It was also registered with Clinicaltrials. gov, identifier: NCT02495194 , three months and six months after the HT intervention group received treatment. The control group started its first session only after the completion of all the above-mentioned biopsychosocial assessments. Thereafter, there were no further assessments on the waitlist control group.g for 25 min at room temperature to obtain the plasma. All the blood and plasma samples from both groups at the three time-points were stored at −80 °C. The nine plasma biomarkers were examined using commercially available enzyme-linked immunosorbent assay (ELISA) kits, namely, IL-6, IL-1β , CXCL12/SDF-1α, CXCL5/RANTES , BDNF , DHEA , cortisol, hs-CRP , and sgp130 . Assays for each biomarker were run on the same day to avoid batch effect.To measure the biomarkers, ten millilitres of fasting venous blood were withdrawn from each participant between 8.30 a.m. and 9.30 a.m. at the research centre. The blood was sent to the laboratory within three hours after the first venepuncture. Subsequently, the whole blood samples were centrifuged at 1650 The Montreal Cognitive Assessment (MoCA) was used to assess the cognitive function of the participants. We used the Zung Self-Rating Depression Scale (SDS), and the Zung Self-Rating Anxiety Scale (SAS) to assess psychopathology and the Ryff’s Scales of Psychological Well-Being, Friendship Scale and Satisfaction with Life Scale to assess the psychosocial well-being of the participants. Detailed descriptions of the measures can be found in our protocol paper .t-test, chi-square or Fisher’s exact tests. A general linear model using repeated-measured analysis of variance (rANOVA) was employed to examine the difference between the active treatment and waitlist control groups. The three time-points were entered as a within-participants factor (indicated as “time”) while the two groups of participants were entered as a between-participants factor (indicated as “group”). When the rANOVA indicated significant interaction between group and time (between-group difference), or a significant main effect of time (within-group difference), post-hoc analyses of paired T-tests were conducted to determine the pair of data that differed significantly. A two-tailed p-value of <0.05 was considered as statistically significant. Alpha values of the paired T-tests were adjusted for using Bonferroni’s correction. Analyses and construction of the graphs and figures were performed using the Statistical Package for the Social Sciences (SPSS) version 23.0  and GraphPad Prism version 6 , respectively. Mean imputation of data was performed to replace loss-to-follow-up missing values, hence enabling intention-to-treat analyses. Log- or squared-transformations were employed for highly skewed data.All data were collected and analysed from 2015 to 2017. The biological and psychosocial measurements were expressed as mean ± standard deviation (SD). Baseline variables were compared using Student’s p > 0.05). p = 0.095) (p = 0.02) in the HT group (There was a trend suggesting that time had a significant effect on IL-6 levels (= 0.095) . Post hoHT group a. There p = 0.007) (p = 0.003) in the waitlist control group (Time had a significant effect on CXCL12 (SDF-1α) levels (= 0.007) . Post hool group c. There p = 0.005) (p = 0.05) in the waitlist control group (Time had a significant effect on CXCL5 (RANTES) levels (= 0.005) . Post hool group d. There p = 0.02) (p = 0.07). A significant decrease in plasma BDNF levels from 3-months to 6-months  in the waitlist control group was also observed (Time had a significant effect on BDNF levels ( = 0.02) . Post hoobserved e. There p = 0.01)  . Post ho = 0.01) i. p < 0.001) (p = 0.001). On the other hand, the change in the control group after 6 months was not significant (p = 0.31), with the mean scores reduced by −0.7 points (95% CI: −2.09 to 0.69). There was a significant time × group interaction on the sub-scale of the Ryff’s Scales of Psychological Well-Being, positive relations with others (< 0.001) . Post hop = 0.004) and SAS scores (p = 0.03). There were no significant interactions between time and study groups nor major effects of time or study group in other psychometric measures (p < 0.001), while in the control group from 25.79 (5.07) to 34.21 (3.69), (p < 0.001) (There were significant effects of time on MoCA (measures . No sign< 0.001) . Using an RTC design, preliminary effects on the biological and psychological benefits of HT were explored. A significant decrease in the mean plasma IL-6 level was observed in the HT group but not in the waitlist control group. On the other hand, significant decreases in plasma CXCL5 (RANTES), CXCL12 (SDF-1α) and BDNF were observed in the waitlist control group but not in the HT group. The changes in these biomarkers suggest that engagement in HT may elicit positive biological changes, such as the reduction of inflammation and the ability to maintain hematopoietic brain support and neuroprotection, hence decreasing the risk of psychiatric morbidities, particularly dementia. Similarly, the analyses showed a significant increase in the mean scores of the psychosocial scale, positive relations with others, in the HT group, but not the control group. There were no significant changes in plasma IL-1β, cortisol, DHEA, hs-CRP and Sgp-130 levels and other psychosocial scales in both the HT and control groups.The significant reduction in plasma IL-6 levels after receiving the HT intervention suggests that HT may attenuate heightened inflammation in older adults. High IL-6 levels have been associated with a variety of inflammation-associated diseases including rheumatoid arthritis , asthma The significant reduction in plasma CXCL12 (SDF-1α) levels in the control group suggests deficient hematopoietic brain support which may lead to the deterioration of blood supply to the brain . CorrespThe significant decline in plasma CXCL5 (RANTES) levels in the control group suggests a deterioration in neuroprotection. It has been proposed that CXCL5 (RANTES) promotes leukocyte infiltration in sites of inflammation and activates T cells . AdditioThe significant decline in BDNF in the control group suggests a deterioration in neuronal protection and survival, axonal and dendritic growth. Low peripheral levels of BDNF is associated with lower cognitive test scores and mild cognitive impairment  and fraiThere were no significant changes in plasma Sgp130 levels in either group. Significant changes in IL-6 levels without changes in Sgp130 levels may indicate that other mechanisms are in place. Additionally, the lack of changes in plasma cortisol levels from baseline to 6-months was not unexpected. In a study of an adult population, elevated cortisol and DHEA levels were observed in response to psychosocial stressors . It is nThis study has addressed a number of the shortcomings in the previous studies on HT. Firstly, this is a RCT comparing the HT treatment group with the waitlist control group, which overcomes the limitations of a single group design . SecondlNonetheless, this RCT has several limitations and we thus propose future actions to address these. Firstly, the main limitation of this RCT is the moderate sample size, which indicates the preliminary nature of the results. Although this RCT provides promising data, there were only within-group differences, with no significant between-group differences. These results could be improved with replications using a larger sample size, a longer study duration and higher frequency. Larger sample size may reduce the standard deviations, thus enabling the detection of significant differences between study groups. Another consideration for future research is the “dosage” of the intervention; a diary could be distributed to each participant to note down the exact hours of engaging in HT in-between sessions. Examining the impact of the “dosage” of the intervention could be valuable in pinpointing the optimum hours of exposure to HT to elicit significant effects. Additionally, due to ethical considerations and the exploratory nature of this study, waitlist control design was employed instead of parallel group design. Thus, we were unable to discern the active component of HT. Positive relationships with others was found to be significantly higher in the HT group after intervention, both in our RCT and in previous studies. Future HT trials could control for social interaction using an active control design to discern the exact biopsychosocial mechanisms of HT. Mechanisms of HT intervention on these novel biomarkers were not examined in this study. Future research on the link between HT and improved biomarkers could be performed, for example, a previous study measured phytoncides released from trees and suggested that they could improve biomarkers . The results of this RCT suggest that HT could potentially be useful for reducing inflammation and protecting neuronal functions in healthy elderly adults by reducing plasma IL-6 levels and maintaining plasma CXCL12 (SDF-1α), CXCL5 (RANTES) and BDNF levels. We have provided preliminary biological evidence for the benefits of HT, as well as some hypothesized mechanisms underlying HT that may improve physical and mental health. The significant reductions in plasma IL-6 levels after 6-months in the HT group points to the possibility that elderly adults with inflammatory diseases associated with high IL-6 levels such as depression, dementia, rheumatoid arthritis and cancer could garner benefits from HT. Furthermore, our data contributes to the knowledge gap on how HT might exert hematopoietic brain support and prevent neurodegeneration by maintaining plasma CXCL12 (SDF-1α), CXCL5 (RANTES) and BDNF levels. Further biological studies on the effects of HT are necessary to replicate these findings in larger elderly populations, particularly those with inflammatory disorders and/or high risk of dementia.


Sapium sebiferum oil-derived dimer acid -non-3-enyl]-10-octylnonadecanedioic acid). Meanwhile, DACC can be used for synthetic materials of bio-based non-isocyanate polyurethane (bio-NIPU). In this study, DACC was synthesized by the esterification of dimer acid and glycerol carbonate using Novozym 435 (Candida antarctica lipase B) as the biocatalyst. Via the optimizing reaction conditions, the highest yield of 76.00% and the lowest acid value of 43.82 mg KOH/g were obtained. The product was confirmed and characterized by Fourier transform-infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Then, the synthetic DACC was further used to synthesize bio-NIPU, which was examined by FTIR, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), indicating that it possesses very good physio-chemical properties and unique material quality with a potential prospect in applications.Dimer acid cyclocarbonate (DACC) is synthesized from glycerol carbonate (GC) and  Sapium sebiferum oil, a non-edible oil, is abundant in China and its production significantly benefits the environment construction, it has been attracting wide attention and has been moderately studied -10-octylnonadecanedioic acid, CAS No. 61788-89-4) and glycerol carbonate  were purchased from Bangcheng Chemical Ltd.  and Tokyo Chemical Industry Co., Ltd. , respectively. Novozym 435 (immobilized lipase B from C. antarctica) with a specific activity 10,000 propyl laurate units (PLUs) per gram was commercially obtained from Novozym Co. Ltd. . Acetonitrile (CAS No. 75-05-8), hexane (CAS No. 110-54-3), dichloromethane (CAS No. 75-09-2), molecular sieve type 4A (CAS No. 70955-01-0), ethylenediamine , diethylenetriamine , triethylenetetramine , tetraethylenepentamine , and hexanediamine  were analytical reagents and bought from Sinopharm Chemical Reagent Ltd. Co. .Dimer acid , temperature (35–65 °C), molecular sieve content , agitation speed (50–300 rpm), solvent , and Novozym 435 cycle number (1–5 times) on the production of DACC were explored. The yield and acid value were chosen as the indicators.DACC was synthesized in acetonitrile via an esterification reaction between DA and GC catalyzed by Novozym 435 ,33,34,35All the esters were purified after being prepared. These purified materials were further used in follow-up experiments to characterize their structures and calculate the conversion rates .n-hexane and the esters precipitated out, aiming to separate DA. Finally, precipitation in deionized water afforded DACC as a yellow viscous liquid; the residual water from the production was evaporated.The enzyme was removed from the reaction mixture via filtration and the solvent was evaporated using a rotovap system. Then, the filtered and evaporated reaction mixtures were dissolved in DACC and dichloromethane were placed in the Teflon mold and stirred mechanically for 5 min. Then, different amines  were added and the mixture was stirred mechanically for 5 min . Thereaf−1 with a spectral resolution of 4 cm−1 using a Bruker Vertex70 FTIR spectrometer equipped with a DTGS detector . Taking the KBr plate as a blank for the background, a few of the samples were dropped on it for the test.The FTIR spectra of the samples were recorded in the frequency range of 4000–400 cm1H-NMR spectra of the samples were determined by a Bruker AV600 MHz NMR spectrometer . The samples were dissolved in CDCl3 (Tetramethylsilane (TMS) as internal standard) and then placed in 5 mm diameter NMR sample tubes for the determination. The measurement was performed at room temperature.The To confirm the degree of esterification, samples were characterized by acid value according to Chinese National Standard (GB/T 264-1983) . The yie2 atmosphere.Thermogravimetric analyses (TGA) were performed using a Pyris 1 TGA  at a heating rate of 10 °C /min. Approximately 5 mg of sample was subjected to temperatures from 50 to 600 °C in an N2 atmosphere. The sample was first heated from −50 to 100 °C at 10 °C/min.Differential scanning calorimetry (DSC) analysis was performed on a Perkin-Elmer Diamond DSC instrument  under NAccording to the chemical equation in To find the proper reaction time, the reaction was performed from 4 to 24 h. As shown in Enzymes are more efficient and environmentally friendly catalysts than chemical catalysts. Here, Novozym 435 was employed to catalyze DACC synthesis. The effect of enzyme concentration and its optimal dosage were investigated at various levels ranging from 1 to 10 wt % c. It canThe reaction temperature exerts a significant effect on enzyme activity and the economic effect of the DACC synthesis . The effBecause hydrolysis is the reverse reaction of esterification, removing water is the key to the degree of esterification. The effect of molecular sieve content was investigated over a range from 30 to 100 wt % e. The reIn this reaction, agitation speed is an easily overlooked single factor. In this reaction, dimer acid and glycerol carbonate do not mix. Therefore, a suitable solvent should dissolve enough raw materials for the lipase-catalyzed esterification, and the solvent should not affect lipase activity and stability. The results show that the reaction had the lowest yield in tetrahydrofuran, which might have altered the native conformation of the lipase g. Tert-bImmobilized lipase has a reusable feature. We performed a series of experiments to examine its characteristic for the synthesis of DACC. After one cycle, the immobilized lipase was recovered by paper filtration and then dried for the next cycle. As shown in −1 attributed to a carboxyl group. The curve below is the FTIR spectra of DACC, which had two carbonyl C=O stretching vibrations attributed to an ester group at 1742.62 cm−1 and carbonic ester at 1810.65 cm−1. Obviously, the carboxyl group disappeared and two new carbonyl C=O stretching vibrations appeared because the carboxyl group reacted with a hydroxyl group to get an ester group. The FTIR spectra of DA and DACC clearly indicate the formation of DACC, especially the changes of the different carbonyl C=O stretching vibrations.2CH–, 2H), δ 4.29–4.34, 4.56–4.59 , δ 4.99–5.03 , suggesting that the DACC had been successfully synthesized.Compared with the spectra of DA a, the sp−1 only existed in DACC because it is the carbonyl C=O stretching vibration from carbonic ester. As can be seen from −1, which are the N–H stretching vibration and C–N stretching vibration, respectively, in bio-NIPU. The results confirm successful synthesis of bio-NIPU by DACC and different amines.Tg) values of the bio-NIPUs with secondary amines were higher than those of the bio-NIPUs with diamine. Tg value of DETA-NIPU was −6.00 °C, which was higher than the other NIPUs. The results suggest that secondary amines have a positive effect on the crystallization of bio-NIPU. These bio-NIPUs have low Tg values due to the soft segment long chain structure of dimer acid. There was only one single glass transition temperature for all bio-NIPUs, indicating good miscibility. To study the thermal properties of different bio-NIPUs, the DSC curves are recorded in Td5% (the temperature at 5% weight loss) around 210 °C. The thermal stability value of TEPA-NIPU was lower with a Td5% equal to 194 °C (Tmax) for each of the two stages for different bio-NIPUs are given in Tmax1 values of DETA-NIPU, TETA-NIPU, and TEPA-NIPU were about 220 °C. However, Tmax1 values of EDA-NIPU and HMDA-NIPU were higher due to no reaction between the secondary amines and cyclocarbonates. The Tmax2 value of TEPA-NIPU was 468 °C, which was the highest temperature in the different bio-NIPUs. This result is due to the high boiling point of TEPA, which may influence the maximum rate of degradation of the soft segments. The influence of the average amine functionality was investigated for the thermal stability of the synthesized polymers via thermogravimetric analysis (TGA). o 194 °C . The chaS. sebiferum oil by esterification using Novozym 435 as the catalyst. Considering the economic effect and efficiency criteria, GC/DA molar ratio, reaction time, Novozym 435 concentration, reaction temperature, molecular sieve content, agitation speed, and different solvents were optimized one by one. Meanwhile, enzyme cycling was studied. The yield was 76.00% and the lowest acid value for synthesized DACC was 43.82 mg KOH/g under optimal conditions. The product was further confirmed by FITR and NMR. Then, DACC was employed to synthesize bio-NIPU and subsequently characterized by FTIR, DSC, and TGA. These studies prove that it is feasible to synthesize DACC by lipase. Furthermore, the bio-NIPU had a low Tg and a good thermostability, which can be potentially used in the coating industry. This study demonstrates a new method to synthesize cyclocarbonate, which has a prosperous future potential for the bio-polyurethane industry.This study attempted to optimize the enzymatic catalyzed production of DACC from DA and GC derived from